The transmissible spongiform encephalopathies (TSE) constitute a group of neurodegenerative diseases. In humans these diseases include Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome, Fatal Familial Insomnia, and Kuru (see, e.g., Harrison""s Principles of Internal Medicine, Isselbacher et al., eds., McGraw-Hill, Inc. New York, (1994); Medori et al. 1992 N. Engl. J. Med., 326: 444-9.). In animals the TSE""s include sheep scrapie, bovine spongiform encephalopathy, transmissible mink encephalopathy, and chronic wasting disease of captive mule deer and elk (Gajdusek, (1990) Subacute Spongiform Encephalopathies: Transmissible Cerebral Amyloidoses Caused by Unconventional Viruses. Pp. 2289-2324 In: Virology, Fields, ed. New York: Raven Press, Ltd.). Transmissible spongiform encephalopathies are characterized by the same hallmarks: a spongiform degeneration, reactive gliosis in the cortical and subcortical gray matters of the brain, and transmission when experimentally inoculated into laboratory animals including primates, rodents, and transgenic mice.
Recently, the rapid spread of bovine spongiform encephalopathy and its correlation with elevated occurrence of spongiform encephalophathies in humans has lead to a significant increase of interest in the detection of transmissible spongiform encephalopathies in non-human mammals. The tragic consequences of accidental transmission of these diseases (see, e.g., Gajdusek, Infectious Amyloids, and Prusiner Prions In Fields Virology. Fields, et al., eds. Lippincott-Ravin, Pub. Philadelphia (1996); Brown et al. (1992) Lancet, 340: 24-27), and the decontamination difficulties (Asher et al. (1986) pages 59-71 In: Laboratory Safety: Principles and Practices, Miller ed. Am. Soc. Microb.), and recent concern about bovine spongiform encephalopathy (British Med. J. 1995; 311: 1415-1421) underlie the urgency of having a diagnostic test that would identify humans and animals with transmissible spongiform encephalopathies.
Definitive premortem diagnosis of these transmissible diseases can only be made histopathologically; however, biopsy of brain tissue is not an ideal method due to risks to animals, patients, and health care personnel. Moreover, lesions can be missed because of the patchy nature of the pathological process.
Measurement of most cerebrospinal fluid (CSF) proteins that have been implicated as pre-mortem markers of Creutzfeldt-Jakob disease have not been very useful diagnostically. These include neuron-specific enolase (NSE) (Jimi et al. (1992) Lancet, 211: 37-46; and Zer et al. (1995) Lancet, 345: 160-9-1610), S-100b protein (Jimi et al., supra), brain-type isozyme of creatine kinase (Jimi et al., supra), GTP binding protein G0 (Jimi et al., supra), ubiquitin (Manaka et al. (1992) Neurosci. Letts. 139: 47-49), and lactic acid (Awerbuch et al. (1985) Internat. J. Neurosci., 42: 1-5).
However, two useful marker proteins, designated proteins 130 and 131, were discovered by two-dimensional electrophoresis (2DE) and silver staining surveys of cerebrospinal fluid (CSF) (see, e.g., Harrington et al. (1986) N. Engl. J. Med., 315: 279-283 and U.S. Pat. No. 4,892,814). These markers were shown to have very high sensitivity (21/21) and specificity (515/520) in the diagnosis of Creutzfeldt-Jacob disease, and this test has been extremely useful in the premortem diagnosis of several difficult cases (see, e.g., Croxson et al. (1988) Neurology 38: 1128-30; Blisard et al. (1990) J. Neurological Sci., 99: 75-81; Marzewski et al., (1988) Neurology, 38: 1131-33; Macario et al. (1991) British Med J. 302: 1149). The only other disease in which these proteins were found was herpes encephalitis which is easily distinguished on clinical presentation. Testing for these markers, however, has required the two-dimensional electrophoresis (2DE) technique, which is cumbersome and time-consuming. Thus, despite the very high correlation of these marker proteins with the disease, the two-dimensional electrophoresis biochemical test has not become practical for clinical use.
This invention provides improved assays for the detection of transmissible spongiform encephalopathies (TSEs) in humans and non-human mammals. The invention is premised, in part, on the surprising discovery that elevated levels of 14-3-3 protein(s) in cerebrospinal fluid are indicative of (highly correlated with) transmissible spongiform encephalopathies (e.g., Creutzfeldt-Jacob disease in humans and bovine spongiform encephalopathy (mad cow disease) in bovines).
In a preferred embodiment this invention provides a method of detecting a transmissible spongiform encephalopathy (TSE) in a human or in a non-human mammal. Preferred transmissible spongiform encephalopathies include Creutzfeldt-Jakob Disease in the case of a human, bovine spongiform encephalopathy (BSE or mad cow disease) in the case of a bovine, and scrapie in the case of sheep.
The methods of this invention involve detecting the presence or absence, or quantifying, a 14-3-3 protein in cerebrospinal fluid of the human or non-human mammal: In the case of a human, detection of the 14-3-3 protein is preferably by use of an immunoassay (e.g., Western Blot assay or Sandwich assay). In the case of a non-human mammal, detection is by any convenient means, preferably a two-dimensional electrophoresis or by use of an immune assay (e.g., Western Blot assay or Sandwich assay). When applied to a human, the method can further comprise determining that the human does not suffer from herpes simplex encephalitis.
Preferred immunoassays use an anti-14-3-3 antibody. The antibody can be polyclonal or monoclonal, with polyclonal antibodies being more preferred. Particularly preferred assay formats include Western Blot assays and antigen capture (e.g., Sandwich) assays.
Definitions
The following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein.
The term xe2x80x9c14-3-3 proteinxe2x80x9d is used herein to refer to members of the 14-3-3 class of proteins as it is commonly known to those of skill in the art (see, e.g., Ichimura et al. (1988) Proc. Nat""l. Acad. Sci. USA 85:7084-7088, Zupan et al. (1992) J. Biol. Chem., 267: 8707-8710; Aitken et al. (1992) Trends. Biochem. Sci., 17: 498-501; Burbelo et al. (1995) Current Biology,5: 95-96; Robinson et al. (1994) Biochem. J. 299: 853-861; Ichimura et al. (1988) Proc. Nat""l. Acad. Sci. USA, 85: 7084-7088; and Morgan et al. (1992) Nature, 355: 833-836). Assays that detect 14-3-3 are intended to detect the level of endogenous (native) 14-3-3 present in subject biological sample (e.g., CSF). However, exogenous 14-3-3 (14-3-3 protein from a source extrinsic to the biological sample) may be added to various assays to provide a label or to compete with the native 14-3-3 in binding to an anti-14-3-3 antibody. One of skill will appreciate that a 14-3-3 mimetic may be used in place of exogenous 14-3-3 in this context. An xe2x80x9c14-3-3 mineticxe2x80x9d, as used herein, refers to a molecule that bears one or more 14-3-3 epitopes such that it is specifically bound by an antibody that specifically binds native 14-3-3.
The phrase xe2x80x9cdiagnostic of a transmissible spongiform encephalopathyxe2x80x9d is used herein to refer to a marker or assay that is indicative of the presence or predicts the ultimate onset of a transmissible spongiform encephalopathy. However, it will be appreciated by one of skill in the art, that all assays exhibit a certain level of false positives and false negatives. Even where a positive result in an assay is not invariably associated with the ultimate onset of the encephalopathy (i.e. where there are some false positives), the result is valuable as it results in more careful monitoring of the patient or animal and the institution of appropriate containment procedures thus reducing risk of infection and transmission through the population. An assay is diagnostic of a transmissible spongiform encephalopathy where detection of the assay marker (e.g. 14-3-3 protein) shows a statistically significant association or correlation with the ultimate manifestation of symptoms of a transmissible spongiform encephalopathy (e.g., Creutzfeldt-Jacob disease in humans or mad cow disease in bovines).
The term xe2x80x9ccerebrospinal fluidxe2x80x9d or xe2x80x9cCSFxe2x80x9d as used herein includes whole cerebrospinal fluid or derivatives or fractions thereof well known to those of skill in the art. Thus a cerebrospinal fluid sample can include various fractionated forms of cerebrospinal fluid or can include various diluents as may be added to facilitate storage or processing in a particular assay. Such diluents are well known to those of skill in the art and include various buffers, preservatives and the like.
As used herein, an xe2x80x9cimmunoassayxe2x80x9d is an assay that utilizes an antibody to specifically bind to the analyte. The immunoassay is characterized by the use of specific binding to a particular antibody as opposed to other physical or chemical properties to isolate, target, and quantify the analyte.
As used herein, an xe2x80x9cantibodyxe2x80x9d refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
The basic immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one xe2x80x9clightxe2x80x9d (about 25 kD) and one xe2x80x9cheavyxe2x80x9d chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VL) refer to these light and heavy chains respectively.
Antibodies may exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)xe2x80x22, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)xe2x80x22 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the F(ab)xe2x80x22 dimer into an Fabxe2x80x2 monomer. The Fabxe2x80x2 monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y. (1993) for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fabxe2x80x2 fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies.
The phrase xe2x80x9cspecifically binds to a proteinxe2x80x9d or xe2x80x9cspecifically immunoreactive withxe2x80x9d, when referring to an antibody refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to a protein under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, antibodies can be raised to the human 14-3-3 protein that bind 14-3-3 and not to any other proteins present in a biological sample (e.g., CSF). A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
A xe2x80x9cconcentration standardxe2x80x9d is a predetermined concentration of a particular moiety, in this case a 14-3-3 protein, that is used for standardizing an assay for that moiety. A negative control, is a sample that lacks any of the specific analyte the assay is designed to detect and thus provides a reference baseline for the assay.